Oligonucleotide inhibitor of dna polymerases

ABSTRACT

The invention comprises a reversible oligonucleotide inhibitor of nucleic acid polymerases. Methods of designing said inhibitors and using said inhibitors in amplification and detection of nucleic acid, particularly detection of RNA by RT-PCR are also disclosed.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 31, 2014, isnamed 31881-US1_SL.txt and is 1,912 bytes in size.

FIELD OF THE INVENTION

The invention relates to the field of nucleic acids amplification andspecifically, to reducing non-specific amplification by a thermostableDNA polymerase via the use of a reversible oligonucleotide inhibitor ofthe polymerase.

BACKGROUND OF THE INVENTION

The Polymerase Chain Reaction (PCR) and real-time PCR have been widelyaccepted in the research and clinical fields as rapid and specificmethods of detecting a target nucleic acid. However, the problem ofnon-specific amplification, i.e., amplification of non-target sequences,is often a limiting factor in achieving high sensitivity and specificityrequired for clinical applications. See Mackay, I. M. (2004) Real-timePCR in the microbiology laboratory, Clin. Microbiol. Infect. 10:190.

The non-specific amplification is thought to result from extension ofprimers annealed to secondary, partially complementary sites in thegenome or to primer cross-annealing or self-annealing. The presence ofnon-specific extension products has been attributed to polymeraseactivity at ambient temperature where such partially complementaryprimer-template duplexes are stable. (Chou et al. (1992) Prevention ofpre-PCR mis-priming and primer dimerization improves low-copy-numberamplifications Nucleic Acid Res. 20:1717). Accordingly, methods ofinhibiting the primer extension activity of the polymerase at ambienttemperature have been devised. These methods termed “hot start” assurethat the polymerase becomes fully active only when the temperature ishigh enough to destabilize non-specific primer-template complexes sothat extension of the primers at non-specific sites is avoided.

One hot start method involves an oligonucleotide that binds and inhibitsthe polymerase at low temperature, but not at high temperature. See Dangand Jayasena (1996) Oligonucleotide inhibitors of Taq DNA polymerasefacilitate detection of low copy number targets by PCR J. Mol. Biol.264:268. These oligonucleotides are extremely specific for and have highaffinity to the target enzyme.

The hot-start approach however is not suitable for reverse transcriptionPCR (RT-PCR) applications where the reverse transcriptase requirestemperatures below 50° C. Certain thermostable DNA polymerases havereverse transcription activity, allowing the use of a single enzyme toperform reverse transcription and amplification of cDNA (RT-PCR) in thesame reaction mixture. See Myers and Gelfand (1991) Reversetranscription and DNA amplification by a Thermus thermophilus DNApolymerase, Biochemistry 30:7661. However, RNA is labile at hightemperature in the presence of divalent ions necessary for polymeraseactivity. For that reason, reverse transcription is carried out at lowertemperature (50-60° C.) prior to commencement of the traditional PCRthermocycling. A typical oligonucleotide aptamer has melting temperatureclose to 60° C. and does not sufficiently release the inhibition of thepolymerase at lower temperatures. It is therefore desirable to obtain areversible polymerase inhibitor that could release inhibition at lowertemperatures.

SUMMARY OF THE INVENTION

In one embodiment, the invention is a reversible inhibitor of nucleicacid polymerases comprising a single-stranded DNA oligonucleotide havingone or more regions of double-stranded secondary structure, wherein atleast one of said regions comprises at least one uracil base. Thedouble-stranded secondary structure may be stable under ambienttemperature in a PCR mixture. In some embodiments, the reversibleinhibitor has SEQ ID NO:1 wherein one or more thymine bases are replacedwith a uracil base, e.g., SEQ ID NOs: 2-4.

In another embodiment, the invention is a method of designing areversible inhibitor of nucleic acid polymerases comprising designing asingle-stranded DNA oligonucleotide having one or more regions ofdouble-stranded secondary structure, wherein at least one of saidregions comprises at least one uracil base. The oligonucleotide may beselected from a mixture of oligonucleotides using systematic evolutionof ligands by exponential enrichment (SELEX).

In yet another embodiment, the invention is a method of reversiblyinhibiting a nucleic acid polymerase in a reaction mixture comprisingcontacting the mixture with a single-stranded DNA oligonucleotide havingone or more regions of double-stranded secondary structure, wherein atleast one of said regions comprises at least one uracil base. The methodmay further comprise contacting the mixture with a uracil-N-glycosylase,optionally in the temperature range of 40-65° C. The method may furthercomprise contacting the sample with a polyamine, for example, apolyamine is selected from spermidine, spermine and trimethylenediamine.

In yet another embodiment, the invention is a method of amplifying atarget nucleic acid comprising prior to amplification, contacting areaction mixture containing the target nucleic acid with asingle-stranded DNA oligonucleotide having one or more regions ofdouble-stranded secondary structure, wherein at least one of saidregions comprises at least one uracil base. The method may furthercomprise prior to amplification, contacting the sample with auracil-N-glycosylase and optionally, with a polyamine, such as forexample, spermidine, spermine or trimethylenediamine.

In yet another embodiment, the invention is a kit for amplifying atarget nucleic acid containing a reversible inhibitor of a nucleic acidpolymerase comprising a single-stranded DNA oligonucleotide having oneor more regions of double-stranded secondary structure, wherein at leastone of said regions comprises at least one uracil base. The kit mayfurther comprise uracil-N-glycosylase and optionally, a polyamine, suchas for example, spermidine, spermine or trimethylenediamine.

In yet another embodiment, the invention is a reaction mixture foramplifying a target nucleic acid containing a reversible inhibitor of anucleic acid polymerase comprising a single-stranded DNA oligonucleotidehaving one or more regions of double-stranded secondary structure,wherein at least one of said regions comprises at least one uracil base.The reaction mixture may further comprise uracil-N-glycosylase andoptionally, a polyamine, such as for example, spermidine, spermine ortrimethylenediamine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the predicted secondary structure of the oligonucleotideSEQ ID NO: 1 and positions where Ts were replaced with Us to form SEQ IDNOs: 2-4.

FIGS. 2-5 show results of primer extension in the presence of SEQ IDNOs: 1-4 at various temperatures.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “nucleic acid,” and “oligonucleotide” refer to targetsequences and probes. The terms are not limited by length and aregeneric to linear polymers of deoxyribonucleotides (single-stranded ordouble-stranded DNA), ribonucleotides (RNA), and any other N-glycosideof a purine or pyrimidine base, including adenosine, guanosine,cytidine, thymidine and uridine and modifications of these bases.

The term “conventional” or “natural” when referring to nucleic acidbases, nucleoside triphosphates, or nucleotides refers to those whichoccur naturally in the polynucleotide being described (i.e., for DNAthese are adenine or dATP, guanine or dGTP, cytosine or dCTP and thymineor dTTP and for RNA, these are adenine or ATP, guanine or GTP, cytosineor CTP and uracil or UTP).

The term “unconventional,” “non-natural,” or “modified” when referringto a nucleic acid base, nucleoside, or nucleotide includes nucleic acidbase, nucleoside, or nucleotide that does not occur in nucleic acidsfound in nature but is a modification, derivation, or analogue ofconventional bases, nucleosides, or nucleotides. For example, dITP, and7-deaza-dGTP do not occur in nucleic acids in nature but are frequentlyutilized in place of dGTP and 7-deaza-dATP can be utilized in place ofdATP in in vitro DNA synthesis reactions, such as sequencing. Certainunconventional nucleotides are modified at the 2′ position of the ribosesugar in comparison to conventional dNTPs. Ribonucleotides areunconventional nucleotides as substrates for DNA polymerases. As usedherein, unconventional nucleotides include, but are not limited to,compounds used as terminators for nucleic acid sequencing. Exemplaryterminator compounds include but are not limited to those compounds thathave a 2′,3′ dideoxy structure and are referred to as dideoxynucleosidetriphosphates, e.g., ddATP, ddTTP, ddCTP and ddGTP. Additional examplesof terminator compounds include 2′-PO₄ analogs of ribonucleotides (see,e.g., U.S. Pat. No. 7,947,817). Other unconventional nucleotides includephosphorothioate dNTPs ([[α]-S]dNTPs), 5′-[α]-borano-dNTPs,[α]-methyl-phosphonate dNTPs, and ribonucleoside triphosphates (rNTPs).Unconventional bases may be labeled with radioactive isotopes such as³²P, ³³P, or ³⁵S; fluorescent labels; chemiluminescent labels;bioluminescent labels; hapten labels such as biotin; or enzyme labelssuch as streptavidin or avidin. Fluorescent labels may include dyes thatare negatively charged, such as dyes of the fluorescein family, or dyesthat are neutral in charge, such as dyes of the rhodamine family, ordyes that are positively charged, such as dyes of the cyanine family.Dyes of the fluorescein family include, e.g., FAM, HEX, TET, JOE, NANand ZOE. Dyes of the rhodamine family include Texas Red, ROX, R110, R6G,and TAMRA. Various dyes or nucleotides labeled with FAM, HEX, TET, JOE,NAN, ZOE, ROX, R110, R6G, Texas Red and TAMRA are marketed byPerkin-Elmer (Boston, Mass.), Applied Biosystems (Foster City, Calif.),or Invitrogen/Molecular Probes (Eugene, Ore.). Dyes of the cyaninefamily include Cy2, Cy3, Cy5, and Cy7 and are marketed by GE HealthcareBiosciences (Pittsburgh, Pa.).

The term “oligonucleotide” refers to a nucleic acid polymer thatincludes at least two nucleic acid monomer units (e.g., nucleotides). Anoligonucleotide typically includes from about six to about 175nucleotides, more typically from about eight to about 75 nucleotides,e.g., about 15, about 20, about 25, about 30, about 35, or morenucleotides). The exact size of an oligonucleotide will depend on manyfactors, including the ultimate function or use of the oligonucleotide.Many methods exist for preparation of oligonucleotides, e.g., isolationof an existing or natural sequence, DNA replication or amplification,reverse transcription, cloning and restriction digestion of appropriatesequences, or direct chemical synthesis by a method such as thephosphotriester method of Narang et al. (Meth. Enzymol. 68:90-99, 1979);the phosphodiester method of Brown et al. (Meth. Enzymol. 68:109-151,1979); the diethylphosphoramidite method of Beaucage et al. (TetrahedronLett. 22:1859-1862, 1981); the triester method of Matteucci et al. (J.Am. Chem. Soc. 103:3185-3191, 1981).

The term “probe” refers to an oligonucleotide that selectivelyhybridizes to a target nucleic acid under suitable conditions.

The terms “target sequence” or “target” refer to a region of a nucleicacid sequence that is to be analyzed.

The term “sample” refers to any composition containing or presumed tocontain nucleic acid. This includes a sample of tissue or fluid isolatedfrom an individual for example, skin, plasma, serum, spinal fluid, lymphfluid, synovial fluid, urine, tears, blood cells, organs, bone marrowand tumors, including the fresh or fresh-frozen tissue andformalin-fixed paraffin embedded tissue (FFPET), and also to samples ofin vitro cultures established from cells taken from an individual, andnucleic acids isolated therefrom.

The term “aptamer” refers to an oligonucleotide that specificallyrecognizes and binds to DNA polymerase, and efficiently inhibits thepolymerase activity as described in U.S. Pat. No. 5,693,502.

The term “mutant,” in the context of DNA polymerases of the presentinvention, means a polypeptide, typically recombinant, that comprisesone or more amino acid substitutions relative to a corresponding,naturally-occurring or unmodified DNA polymerase.

The term “thermostable polymerase,” refers to an enzyme that is stableat elevated temperatures, is heat resistant, and retains sufficientactivity to effect subsequent polynucleotide extension reactions anddoes not become irreversibly denatured (inactivated) when subjected tothe elevated temperatures for the time necessary to effect denaturationof double-stranded nucleic acids. Thermostable DNA polymerases fromthermophilic bacteria include, e.g., DNA polymerases from Thermotogamaritima, Thermus aquaticus, Thermus thermophilus, Thermus flavus,Thermus filiformis, Thermus species Sps17, Thermus species Z05, Thermuscaldophilus, Bacillus caldotenax, Thermotoga neopolitana, andThermosipho africanus.

The term “thermoactive” refers to an enzyme that maintains its catalyticproperties at temperatures commonly used for annealing and extensionsteps in PCR reactions (i.e., 45-80° C.). Thermoactive enzymes may ormay not be thermostable. Thermoactive DNA polymerases can be DNA or RNAdependent from thermophilic species or from mesophilic speciesincluding, but not limited to, Escherichia coli, Moloney murine leukemiaviruses, and Avian myoblastosis virus.

In the context of DNA polymerases, “correspondence” to another sequence(e.g., regions, fragments, nucleotides or amino acid positions in thesequence) is based on the convention of numbering according tonucleotide or amino acid position and then aligning the sequences in amanner that maximizes the percentage of sequence identity. Because notall positions within a given “corresponding region” need be identical,non-matching positions within a corresponding region may be regarded as“corresponding positions.” Accordingly, as used herein, referral to an“amino acid position corresponding to amino acid position [X]” of aspecified DNA polymerase refers to equivalent positions, based onalignment, in other DNA polymerases and polymerase families.

The terms “identical” or “identity,” or percent identity in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same. Sequences are“substantially identical” to each other if they have a specifiedpercentage of nucleotides or amino acid residues that are the same(e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% identity over aspecified region), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms or by manual alignment andvisual inspection. These definitions also refer to the complement of atest sequence.

The terms “similarity” or “percent similarity,” in the context of two ormore polypeptide sequences, refer to two or more sequences orsubsequences that have a specified percentage of amino acid residuesthat are either the same or similar as defined by a conservative aminoacid substitutions (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%,85%, 90%, or 95% similar over a specified region), when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection.Sequences are “substantially similar” to each other if they are at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, or at least 55% similar to each other.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, for example, by the local homologyalgorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by thehomology alignment algorithm of Needleman and Wunsch (J. Mol. Biol.48:443, 1970), by the search for similarity method of Pearson and Lipman(Proc. Natl. Acad. Sci. USA 85:2444, 1988), or by computerizedimplementations of these algorithms e.g., BLAST, BLASTN, GAP, BESTFIT,FASTA, and TFASTA, or by manual alignment and visual inspection (see,e.g., Ausubel et al., Current Protocols in Molecular Biology (1995supplement)).

The terms “C_(p) value” or “crossing point” value, or “C_(t) value” or“threshold cycle” value are used interchangeably to refers to a valuethat allows quantification of input target nucleic acids. The C_(t)value can be determined e.g., according to the second-derivative maximummethod, see Van Luu-The, et al., (2005) Improved real-time RT-PCR methodfor high-throughput measurements using second derivative calculation anddouble correction, BioTechniques, 38:287.

The term “PCR efficiency” refers to an indication of cycle to cycleamplification efficiency for the perfectly matched primer template. PCRefficiency is calculated for each condition using the equation: % PCRefficiency=(10^((−slope))−1)×100, wherein the slope was calculated bylinear regression with the log copy number plotted on the y-axis andC_(t) plotted on the x-axis.

The present invention comprises a reversible inhibitor of DNApolymerases having the form of an oligonucleotide. Specifically, theoligonucleotide is a DNA oligonucleotide (i.e., composed ofdeoxyribonucleotides) including one or more deoxyuridine (dU)nucleotides (dU-containing inhibitor oligonucleotide). The sequence ofthe oligonucleotide enables the formation of a secondary structurecomprising one or more regions of double stranded secondary structure.The oligonucleotide forms a stable secondary structure under ambienttemperature conditions in the typical reaction mixture, e.g., a PCRmixture. According to the invention, the one or more uracils arepositioned within the regions of the double stranded secondarystructure. The inhibitory properties of the oligonucleotide of thepresent invention are not dependent on the exact nucleotide sequence,but rather on the conformation or shape of the secondary structureformed by the sequence and the melting temperature (T_(m)) of thatstructure. At a temperature below the T_(m), e.g., at ambienttemperature in a typical reaction mixture, the shape assumed by theoligonucleotide allows it to form the oligonucleotide-enzyme complex.The oligonucleotide reversibly inhibits the polymerase enzyme whileassociated with the enzyme in an oligonucleotide-enzyme complex.

It has been shown that introducing non-natural nucleotide can affect theformation and T_(m) (and hence stability) of the secondary structureformed by the oligonucleotide and thus its inhibitory properties. Tothat end, the DNA oligonucleotides have been modified to containribonucleotides, nucleotide analogs, nucleotides with unconventionalbases, non-nucleotide linkers or combinations thereof. (See e.g., U.S.Pat. No. 6,183,679 for description of such non-natural nucleotides ininhibitor oligonucleotides). One of skill in the art can design aninhibitor oligonucleotide (including a dU-containing inhibitoroligonucleotide of the present invention) with a melting temperature andshape suitable for a particular enzyme.

In some embodiments, the dU-containing inhibitor oligonucleotide isdesigned by replacing one or more thymines with uracils in the existingaptamer NTQ21-46A (also referred to as U0)

(SEQ ID NO: 1) 5′-CGATCATCTCAGAACATTCTTAGCGTTTTGTTCTTGTGTATGATC G-3′.In variations of this embodiment, the dU-containing inhibitoroligonucleotide is one of the following sequences:

U1 (SEQ ID NO: 2) 5′-CGAUCAUCTCAGAACATTCTTAGCGTTTTGTTCTTGTGTAUGAUC G-3′U2 (SEQ ID NO: 3) 5′-CGATCATCTCAGAACATTCTTAGCGTTTUGUUCUUGTGTATGATC G-3′U3 (SEQ ID NO: 4) 5′-CGAUCAUCTCAGAACATTCTTAGCGTTTUGUUCUUGTGTAUGAUC G-3′.

Predicted secondary structure of the aptamer NTQ21-46A (U0) and U1, U2and U3 is shown in FIG. 1.

One of skill in the art can easily design similar dU-containinginhibitor oligonucleotides by replacing thymines with uracils or byintroducing uracils in any other positions within the oligonucleotide,e.g., in SEQ ID NO: 1 or in a sequence of another known or novelinhibitor oligonucleotide.

The examples below describe application of the method of the presentinvention to reversibly inhibiting a DNA polymerase from the Thermusspecies Z05 (disclosed in International Application Pub. No.WO1992/06200). However, the method is equally applicable to other DNApolymerases. Analysis of X-ray crystal structures has revealed that DNApolymerases from various organisms fold into similar three dimensionalstructures. The overall folding pattern of DNA polymerases resembles thehuman right hand and contains three distinct subdomains termed “palm,”“fingers,” and “thumb.” (See Beese et al., (1993) Structure of DNApolymerase I Klenow fragment bound to duplex DNA Science 260:352; Patelet al., (1995) Insights into DNA polymerization mechanisms fromstructure and function analysis of HIV-1 reverse transcriptaseBiochemistry 34:5351). While the structure of the fingers and thumbsubdomains vary greatly between polymerases that differ in size and incellular functions, the catalytic palm subdomains are allsuperimposable. For example, motif A, which interacts with the incomingdNTP and stabilizes the transition state during chemical catalysis, issuperimposable with a mean deviation of about one Å amongst mammalianpol α and prokaryotic pol I family DNA polymerases (Wang et al., (1997)Crystal structure of a pol alpha family replication DNA polymerase frombacteriophage RB69 Cell 89:1087. The primary amino acid sequence of DNApolymerase active sites is exceptionally conserved. In the case of motifA, for example, the sequence DYSQIELR (SEQ ID NO: 6) is retained inpolymerases from organisms separated by many millions years ofevolution, including, e.g., Thermus aquaticus, Chlamydia trachomatis andEscherichia coli.

In some embodiments, the U-containing inhibitor oligonucleotideaccording to the present invention can be designed and oligonucleotidesequences disclosed here can be used and further optimized to similarlyreversibly inhibit other thermostable or thermoactive DNA polymerases,e.g. DNA polymerases from Thermotoga maritima, Thermus aquaticus,Thermus thermophilus, Thermus flavus, Thermus filiformis, Thermusspecies Sps17, Thermus caldophilus, Bacillus caldotenax, Thermotoganeopolitana, and Thermosipho africanus or similar DNA polymerases atleast 95% identical thereto.

More generally, the U-containing inhibitor oligonucleotide according tothe present invention can be designed to similarly reversibly inhibitother thermostable or thermoactive enzymes such as ligases, helicasesand nucleases (including nuclease activities of DNA polymerases).Reversible oligonucleotide inhibitors for such enzymes have beendescribed, see, e.g., U.S. Pat. No. 8,470,531. These oligonucleotideinhibitors can be changed into the U-containing oligonucleotideinhibitors of the present invention to benefits from the improvedproperties described herein.

In some embodiments, the invention is a method of designing a reversibleinhibitor of nucleic acid polymerases comprising designing asingle-stranded DNA oligonucleotide having one or more regions ofdouble-stranded secondary structure, wherein at least one of saidregions comprises at least one uracil base.

The U-containing inhibitor oligonucleotide of the present invention canbe designed and optimized using an in vitro selection proceduresystematic evolution of ligands by exponential enrichment (SELEX)described in the U.S. Pat. No. 6,183,679. Briefly, in the SELEX method,a mixture of oligonucleotides with different sequences is contacted withthe target, e.g., an enzyme. The mixture of oligonucleotides that bindthe target are partitioned, amplified and subjected to another round ofselection until a small number of oligonucleotides with maximum affinityto the target are identified.

The U-containing inhibitor oligonucleotide of the present invention canbe prepared by any suitable method, for example by direct chemicalsynthesis by the phosphotriester method of Narang et al. (1979) Meth.Enzymol. 68:90-99; the phosphodiester method of Brown et al. (1979)Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucageet al. (1981) Tetrahedron Lett. 22:1859-1862; the triester method ofMatteucci et al., (1981) J. Am. Chem. Soc. 103:3185-3191; any one of theautomated synthesis methods; or the solid support method of U.S. Pat.No. 4,458,066.

In some embodiments, the 3′-end of the U-containing inhibitoroligonucleotide is blocked to prevent extension by the DNA polymerase.In some embodiments, a blocking group (a chemical moiety) is added tothe terminal 3′-OH or 2′-OH in the oligonucleotide. Some non-limitingexamples of blocking groups include an alkyl group, non-nucleotidelinkers, phosphate group, phosphothioate group, alkane-diol moieties oramino group. In other examples, the 3′-hydroxyl group is modified bysubstitution with hydrogen for fluorine or by formation of an ester,amide, sulfate or glycoside. In yet another example, the 3′-OH group isreplaced with hydrogen (to form a dideoxynucleotide).

In other embodiments, the invention is a method of modulating inhibitionof the DNA polymerase by the dU-containing inhibitor oligonucleotide.The oligonucleotide of the present invention contains uracils. Thepresence of uracils in the DNA sequence makes the oligonucleotide atarget for uracil DNA glycosylases. These enzymes recognize uracilspresent in single-stranded or double-stranded DNA and cleave theN-glycosidic bond between the uracil base and the deoxyribose, leavingan abasic site. See e.g. U.S. Pat. No. 6,713,294. Uracil-DNAglycosylases, abbreviated as “UDG” or “UNG” include UNG (EC 3.2.2.3),mitochondrial UNG1, nuclear UNG2, SMUG1 (single strand-selectiveuracil-DNA glycosylase), TDG (TU mismatch DNA glycosylase), MBD4(uracil-DNA glycosylase with a methyl binding domain) and othereukaryotic and prokaryotic enzymes (See Krokan H. E. et al. (2002)Uracil in DNA—occurrence, consequences and repair, Oncogene21:8935-9232).

Therefore according to the method of the invention, the reaction mixtureno longer needs to be heated to the temperature exceeding the T_(m) ofthe inhibitory oligonucleotide in order to unravel its secondarystructure and release the inhibition of the enzyme in theoligonucleotide-enzyme complex. Instead, the reaction mixture containingthe U-containing inhibitor oligonucleotide of the present invention iscontacted by one of the uracil-N-glycosylases such as for example, UNG.Conveniently, UNG is active in a standard PCR reaction mixture. Thisenables adding UNG to assembled PCR reactions or even to the PCR mastermix. Before the start of thermal cycling, the reaction mixture isincubated at a temperature optimal for the UNG activity within thecontext of the PCR master mix (about 50° C.) or within the temperaturerange where UNG is active. UNG will cleave off the uracil in thedU-containing inhibitor oligonucleotide leaving an abasic site. Thebackbone of DNA with abasic sites is known to be labile, especially athigh temperature under high pH conditions. According to the invention,the one or more uracils and hence one or more abasic sites resultingfrom the UNG cleavage are located in the regions of the double strandedsecondary structure, said structure will unravel upon cleavage andsubsequent backbone cleavage.

In some embodiments, the method further comprises contacting thereaction mixture with a polyamine such for example, spermidine, spermineor trimethylenediamine as described in U.S. application Ser. No.12/485,569 filed on Jun. 16, 2009. In some embodiments the polyamine isan intercalator amine. Polyamines of this group possess an intercalatingmoiety, capable of intercalating between the base pairs or bases in anucleic acid. Examples of intercalating moieties on a polyamine includearenes and polyarenes, such as naphthalene and anthraquinone. In someembodiments, the intercalator moiety itself may also be substituted withone or more polyamine side chains. The addition of polyamines has beenshown to facilitate degradation of abasic DNA resulting from UNGcleavage. Specifically at 50° C., the degradation is improved by1000-fold.

According to the method of the invention, the U-containing inhibitoroligonucleotide can switch from stable secondary structure to unraveledstructure at a lower temperature than oligonucleotide inhibitorsdescribed previously. This feature is especially beneficial for reactionmixtures and methods involving RNA templates that are labile in atypical reaction mixture under the temperature needed to release theenzyme inhibition. Release of the inhibition at lower temperatures willincrease the efficiency of the reverse transcription step of RT-PCR byincreasing the amount of available template.

In general, the U-containing inhibitor oligonucleotide of the presentinvention may be used in any method in which reversible inhibition of aDNA polymerase is desired. For example, the oligonucleotide can be usedin DNA sequencing, DNA or RNA amplification, reverse transcription,reverse transcription PCR (RT-PCR), or primer extension, e.g., indetecting single nucleotide polymorphisms (SNPs) by single nucleotideprimer extension.

In some embodiments, the invention is a method of amplification andoptionally detection of a target nucleic acid sequence comprisingcontacting the sample in a reaction mixture (optionally a PCR reactionmixture containing all the components of PCR except the DNA polymerase)with a DNA polymerase and a reversible DNA-polymerase inhibitor in theform of a U-containing inhibitor oligonucleotide. In variations of thisembodiment, the method further comprises contacting and incubating thereaction mixture with uracil-N-glycosylase enzyme. In variations of thisembodiment, the method further comprises simultaneously of subsequentlyincubating the reaction mixture with a polyamine, optionally selectedfrom spermidine, spermine or trimethylenediamine. The incubation maytake place at 40-65° C., or any one of the 40, 45, 50, 55, 60 or 65° C.or any temperature in between, e.g., at 50° C. The method furthercomprises amplification and optionally detection of the target nucleicacid by PCR.

In yet another embodiment, the invention is a reaction mixture foramplifying a target nucleic acid containing a reversible inhibitor of anucleic acid polymerase comprising the U-containing inhibitoroligonucleotide of the present invention which is a single-stranded DNAoligonucleotide having one or more regions of double-stranded secondarystructure, wherein at least one of said regions comprises at least oneuracil base. In variations of this embodiment, the reaction mixturefurther contains uracil-N-glycosylase, such as UNG. In other variationsof this embodiment, the reaction mixture further contains a polyamine,optionally selected from spermidine, spermine or trimethylenediamine. Infurther variations of this embodiment, the kit further comprisesreagents for PCR or RT-PCR including without limitation, nucleic acidprecursors (dNTPs or NTPs), the polymerase enzyme, oligonucleotides(primers and optionally, probes) and buffers suitable to support theactivity of the enzyme. In some embodiments, the target nucleic acid isRNA. In some embodiments, the polymerase is selected from DNApolymerases from Thermotoga maritima, Thermus aquaticus, Thermusthermophilus, Thermus flavus, Thermus filiformis, Thermus species Sps17,Thermus species Z05, Thermus caldophilus, Bacillus caldotenax,Thermotoga neopolitana, and Thermosipho africanus. In some embodiments,the polymerase is the DNA polymerase from Thermus species Z05 or Thermusthermophilus.

In yet another embodiment, the invention is a kit for amplifying atarget nucleic acid containing a reversible inhibitor of a nucleic acidpolymerase comprising the U-containing inhibitor oligonucleotide of thepresent invention which is a single-stranded DNA oligonucleotide havingone or more regions of double-stranded secondary structure, wherein atleast one of said regions comprises at least one uracil base. Invariations of this embodiment, the kit further containsuracil-N-glycosylase, such as UNG. In further variations of thisembodiment, the kit further contains a polyamine, optionally selectedfrom spermidine, spermine or trimethylenediamine. In further variationsof this embodiment, the kit further comprises reagents for PCR or RT-PCRincluding without limitation, nucleic acid precursors (dNTPs or NTPs),the polymerase enzyme, oligonucleotides (primers and optionally, probes)and buffers suitable to support the activity of the enzyme. In someembodiments, the polymerase is selected from DNA polymerases fromThermotoga maritima, Thermus aquaticus, Thermus thermophilus, Thermusflavus, Thermus filiformis, Thermus species Sps17, Thermus species Z05,Thermus caldophilus, Bacillus caldotenax, Thermotoga neopolitana, andThermosipho africanus. In some embodiments, the polymerase is the DNApolymerase from Thermus species Z05 or Thermus thermophilus.

Examples Example 1 Design of the U-Containing Inhibitor Oligonucleotides(U-Aptamers)

The existing aptamer NTQ21-46A (U0)

(SEQ ID NO: 1) 5′-CGATCATCTCAGAACATTCTTAGCGTTTTGTTCTTGTGTATGATC G-3′was modified by replacing dTs with dUs to create the followingU-aptamers:

U1 (SEQ ID NO: 2) 5′-CGAUCAUCTCAGAACATTCTTAGCGTTTTGTTCTTGTGTAUGAUC G-3′U2 (SEQ ID NO: 3) 5′-CGATCATCTCAGAACATTCTTAGCGTTTUGUUCUUGTGTATGATC G-3′U3 (SEQ ID NO: 4) 5′-CGAUCAUCTCAGAACATTCTTAGCGTTTUGUUCUUGTGTAUGAUC G-3′The predicted secondary structure of the aptamers is shown on FIG. 1.

Example 2 Determination of the Melting Temperature of the U-Aptamers

Melting temperature of the U0, U1, U2 and U3 aptamers (SEQ ID NOs: 1, 2,3 and 4) was determined in a reaction mixture containing 50 mm TrisHCl,pH 8.0, 100 mM KCl, 1 mM dNTPs, 2.5 mM MgCl₂, SYBR® Green I (MolecularProbes (Life Technologies, Inc.) Carlsbad, Calif.) at 0.5×, 20 nM DNApolymerase Z05-D (where indicated) and 0.2 μM of one of the aptamers.Melting curve analysis was performed in LightCycler® 480 (RocheMolecular Diagnostics, Indianapolis, Ind.) according to themanufacturer's instructions. The results are shown in Table 1. Theresults demonstrate that replacing Ts with Us lowers the meltingtemperature of the oligonucleotide secondary structure.

TABLE 1 Melting temperatures of aptamers T_(m), ° C. Aptamer dU contentZ05D+ Z05D− U1 Low 58.4 56.8 U2 Medium 59.5 57.9 U3 High 57.6 55.6 U0None 60.7 59.0

Example 3 Determination of the Melting Temperature of the U-Aptamers inthe Presence of UNG

Melting temperature of the U0, U1, U2 and U3 aptamers (SEQ ID NOs: 1, 2,3 and 4) was determined in a reaction mixture described in Example 1,except where indicated, 0.5 U/μL of UNG was added. To allow for UNGcleavage, all reaction mixtures were incubated at 37° C. prior to themelting curve analysis. The results are shown in Table 2. The resultsdemonstrate that replacing Ts with Us and subsequent cleavage with UNGsubstantially lowers the melting temperature of the oligonucleotidesecondary structure.

TABLE 2 Melting temperatures of aptamers after cleavage with UNG Tm, °C. dU Z05D−/ Z05D−/ ZO5D+/ UNG Aptamer content UNG− UNG+ UNG+ ΔT_(m) U1Low 58.4 52.6 52.6 5.8 U2 Medium 59.2 52.5 52.5 6.7 U3 High 56.7 38.538.6 18.2 U0 None 60.6 60.4 60.5 0.2

Example 4 Primer Extension in the Presence of U-Aptamers and UNG atDifferent Temperatures

The DNA polymerase activity in the presence of oligonucleotideinhibitors was determined by primer extension in the presence of variousconcentrations of the U0, U1, U2 or U3 aptamers (SEQ ID NOs: 1, 2, 3 and4). The assay was performed using M13 mp18 single-stranded DNA (M13;GenBank Accession No. X02513), primed with an oligonucleotide having thefollowing sequence (SEQ ID NO: 5):

5′-GCGCTAGGGCGCTGGCAAGTGTAGCGGTCAC-3′Reactions were initiated by the addition of 12.5 μL of MgCl₂ to 12.5 μLof reaction master mix containing 1 nM of primed M13 template in 96-wellPCR plates. Extension of the primed template was monitored every 6seconds for 99 cycles on the LightCycler′ 480 thermal cycler at thetemperatures indicated. Master mixes contained 2.5 mM MgCl₂, 50 mM TrispH 8.0, 100 mM KCl, a mixture of all four dNTPs, 20 nM of the Z05-D DNApolymerase and SYBR® Green I (Life Technologies, Carlsbad, Calif.) at0.5×, which allowed for the fluorescent detection of primer strandextension. The concentration range of aptamers tested was 0, 50, 200,2000 nM, to ensure that aptamers were present in a 0, 2.5, 10, or 100×molar excess relative to the Z05-D DNA polymerase (see figure legends onFIGS. 2-5). The DNA polymerase activity was quantified by determiningthe slope of increasing fluorescence over time in a linear range. Therelative activities were calculated by normalizing to the average slopeof all reactions conducted in the absence of aptamer at eachtemperature. The results are shown in FIGS. 2-5. The results demonstratethat the addition of UNG drastically reduces aptamer inhibition of theDNA polymerase, particularly for U3.

Example 5 Real Time PCR Amplification of KRAS Codon 12 Target in thePresence of U-Aptamers and UNG

In this example, amplification of the KRAS target in human DNA isperformed in the presence of various concentrations of the U0, U1, U2 orU3 aptamers (SEQ ID NOs: 1, 2, 3 and 4). The nature of the KRAS targetmakes it especially vulnerable to primer dimerization and non-specificamplification. In the absence of hot start, the non-specificamplification obscures the differences in the target concentration andprecludes quantitative analysis. In this example, serial ten-folddilutions of the KRAS target were added to assess quantitative range andspecificity of the method. Amplification was performed in a reactionmixture containing 3 mM MgCl₂, 50 mM Tricine pH 8.0, 55 mM potassiumacetate, 200 μM each dATP, dCTP and dGTP, 300 μM dUTP, 30 μM dTTP, 20 nMof the Z05 DNA polymerase and SYBR® Green I (Life Technologies,Carlsbad, Calif.) at 0.2×, and UNG where indicated. The temperatureprofile in the LightCycler instrument was 50 cycles of 95° C. 15 sec,50, 55 or 60° C. 40 seconds. Amplification was detected by measuringC_(t) values in the presence of various concentrations of the U0, U1, U2or U3 aptamers (SEQ ID NOs: 1, 2, 3 and 4). The concentration range ofaptamers tested was 0, 200, 1000, and 2000 nM, to ensure that aptamerswere present in a 0, 10, 100, or 200× molar excess relative to the Z05DNA polymerase. The results are shown in Tables 3-6.

TABLE 3 Amplification of the KRAS target (C_(t)) without UNG, 60° C. 10xexcess aptamer Target Aptamer copy # none U0 U1 U2 U3 10⁴ 24.5 25.1 25.125.0 25.0 10³ 25.6 28.4 28.3 28.1 28.2 10² 25.8 31.1 30.8 30.6 30.3 none25.7 32.0 31.7 31.2 30.8

TABLE 4 Amplification of the KRAS target (C_(t)) without UNG, 60° C.100x, 200x excess aptamer Target Aptamer Aptamer No Aptamer Copy # 100x200x Aptamer U0 10⁴ 32.8 33.1 10³ 29.2 29.4 10² 25.7 26.0 none 37.7 38.7U1 10⁴ 32.6 32.7 10³ 28.9 29.0 10² 25.6 25.7 none 37.2 38.3 U2 10⁴ 32.432.5 10³ 28.9 29.1 10² 25.7 25.6 none 36.6 38.0 U3 10⁴ 32.0 31.6 10³28.7 28.9 10² 25.4 25.6 none 36.4 37.3 none 10⁴ 26.3 10³ 26.1 10² 24.7none 26.2

The results in Tables 3-4 demonstrate that the aptamers are required todetermine concentration based on C_(t) values and at 60° C. in theabsence of UNG, the U-containing aptamers (SEQ ID NOs: 2, 3 and 4)behave similarly to the parent aptamer (SEQ ID NO: 1).

TABLE 5 Amplification of the KRAS target (C_(t)), 55° C. Aptamer TargetCopy # UNG− UNG+ U0 10² 33.9 39.7 10³ 30.4 35.8 10⁴ 26.9 32.5 None 39.943.3 U1 10² 33.3 35.8 10³ 29.6 32.8 10⁴ 26.1 29.2 None 39.9 38.7 U2 10²33.4 35.5 10³ 29.8 32.5 10⁴ 26.3 28.8 None 39.5 38.0 U3 10² 32.6 34.810³ 29.1 31.8 10⁴ 25.7 28.1 None 37.6 36.0 None 10² 25.9 27.6 10³ 25.827.3 10⁴ 24.6 27.0 None 25.9 27.4

TABLE 6 Amplification of the KRAS target (C_(t)) 50° C. 200x excessaptamer Aptamer Target Copy # UNG− UNG+ U0 10² 35.3 0.0 10³ 31.6 36.910⁴ 28.0 33.7 None 44.3 41.2 U1 10² 34.9 35.8 10³ 30.8 32.4 10⁴ 27.428.8 None 44.7 44.4 U2 10² 34.6 35.2 10³ 30.9 31.9 10⁴ 27.6 28.0 None41.3 39.1 U3 10² 33.9 34.0 10³ 30.3 30.5 10⁴ 26.7 26.9 None 40.2 38.7None 10² 26.9 28.7 10³ 26.6 28.6 10⁴ 25.0 27.5 None 26.9 28.8

The results in Tables 5-6 demonstrate that at lower annealingtemperatures and increased aptamer concentrations, the addition of UNGdrastically reduces dU-containing aptamer inhibition of the DNApolymerase, particularly for U3, as demonstrated by lower C_(t) values.

Example 6 RT PCR Amplification of RNA in the Presence of U-Aptamers andUNG

In this example, amplification of an RNA target (HCV JP2-5, 1000 copiesper reaction) was performed in the presence of various concentrations ofthe U0, U1, U2 or U3 aptamers (SEQ ID NOs: 1, 2, 3 and 4) in thereaction mixture containing UNG as described in Example 5 under thestandard PCR conditions. The concentration range of aptamers tested wasnone, 100-fold and 200-fold molar excess relative to the Z05 DNApolymerase. The results are shown in Table 7.

TABLE 7 Amplification of the HCV RNA target (C_(t)) at 55° C. Foldexcess of Aptamer aptamer to enzyme C_(t) U0 10 ND ND 100 ND ND 200 NDND U1 10 33.5 33.7 100 ND. ND 200 ND ND U2 10 32.8 32.48 100 32.8 32.57200 35.66 35.85 U3 10 32.62 32.57 100 32.48 32.16 200 33.32 33.13 ND =not detected

The results in Table 7 demonstrate that at lower annealing temperatures(e.g., 55° C.) the dU-containing aptamers in the presence of UNG havereduced inhibition of the DNA polymerase, as demonstrated by lower C_(t)values.

While the invention has been described in detail with reference tospecific examples, it will be apparent to one skilled in the art thatvarious modifications can be made within the scope of this invention.Thus the scope of the invention should not be limited by the examplesdescribed herein, but by the claims presented below.

We claim:
 1. A reversible inhibitor of nucleic acid polymerasescomprising a single-stranded DNA oligonucleotide having one or moreregions of double-stranded secondary structure, wherein at least one ofsaid regions comprises at least one uracil base.
 2. The reversibleinhibitor of claim 1 wherein said double-stranded secondary structure isstable under ambient temperature in a PCR mixture.
 3. The reversibleinhibitor of claim 1 comprising SEQ ID NO:1 wherein one or more thyminebases are replaced with a uracil base.
 4. The reversible inhibitor ofclaim 1 selected from SEQ ID NOs: 2-4.
 5. A method of designing areversible inhibitor of nucleic acid polymerases comprising designing asingle-stranded DNA oligonucleotide having one or more regions ofdouble-stranded secondary structure, wherein at least one of saidregions comprises at least one uracil base.
 6. The method of claim 5wherein in the DNA oligonucleotide is selected from a mixture ofoligonucleotides using systematic evolution of ligands by exponentialenrichment (SELEX).
 7. A method of reversibly inhibiting a nucleic acidpolymerase in a reaction mixture comprising contacting the mixture witha single-stranded DNA oligonucleotide having one or more regions ofdouble-stranded secondary structure, wherein at least one of saidregions comprises at least one uracil base.
 8. The method of claim 7,further comprising contacting the mixture with a uracil-N-glycosylase.9. The method of claim 8, wherein the contacting takes place in thetemperature range of 40-65° C.
 10. The method of claim 7, furthercomprising contacting the sample with a polyamine.
 11. The method ofclaim 10, wherein the polyamine is selected from spermidine, spermineand trimethylenediamine.
 12. A method of amplifying a target nucleicacid comprising prior to amplification, contacting a reaction mixturecontaining the target nucleic acid with a single-stranded DNAoligonucleotide having one or more regions of double-stranded secondarystructure, wherein at least one of said regions comprises at least oneuracil base.
 13. The method of claim 12, further comprising prior toamplification, contacting the sample with a uracil-N-glycosylase. 14.The method of claim 12, further comprising contacting the sample with apolyamine.
 15. The method of claim 14, wherein the polyamine is selectedfrom spermidine, spermine and trimethylenediamine.
 16. A kit foramplifying a target nucleic acid containing a reversible inhibitor of anucleic acid polymerase comprising a single-stranded DNA oligonucleotidehaving one or more regions of double-stranded secondary structure,wherein at least one of said regions comprises at least one uracil base.17. The kit of claim 16, further comprising uracil-N-glycosylase. 18.The kit of claim 16, further comprising a polyamine, optionally selectedfrom spermidine, spermine and trimethylenediamine.
 19. A reactionmixture for amplifying a target nucleic acid containing a reversibleinhibitor of a nucleic acid polymerase comprising a single-stranded DNAoligonucleotide having one or more regions of double-stranded secondarystructure, wherein at least one of said regions comprises at least oneuracil base.
 20. The reaction mixture of claim 19, further comprisinguracil-N-glycosylase.
 21. The reaction mixture of claim 20, furthercomprising a polyamine, optionally selected from spermidine, spermineand trimethylenediamine.